Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Labex User Meeting November 2012
Content of this presentation
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History of the D
Clinical aspects
Method and Kit content Free DNA Fetal Kit® RhD
RHD and RHDΨ
Interpretation
Internal Fetal DNA Control Marker
Results from Evaluations and Studies
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IMMUNOHEMATOLOGY
Ancestral Gallery of RhD
Karl Landsteiner
1868 – 1943
Bio-Rad Laboratories
Philip Levine
1900 – 1987
IMMUNOHEMATOLOGY
Alexander Solomon Wiener
1907 – 1976
History
 Wiener and Landsteiner discovered the Rh factor in 1937/1940
 The importance of the Rh factor was the better blood finger print
for criminal matters
 M, N, or P factors where known and Rh factor was just an
additional one
 Later it was recognized that the new Rh factor was associated
with problem in transfusions
 Between 1940 to 1946 Philip Levine discovered the close
association between RhD factor and HDN
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History
 Transfusion for newborn with “Rh disease” saved the babies live
 With this method more than 200.000 lives could be saved
 1961 Finn et al. demonstrated that the administration of anti-D
accelerates the clearance of Rh+ RBC’s
 1965 the first post partum administration of anti-D was given to a
mother.
 1977 the ante- post partum anti-D prophylaxis lead to almost
100% protection of the HDN due to anti-D
 1997 the first publication about cell free fetal DNA by Lo et. al.
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IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Clinical Aspects
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IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
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The test detects fetal RHD gene sequences in maternal
plasma
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The plasma of pregnant woman contains in the course of
the pregnancy an increasing concentration of cell free fetal
DNA
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beginning with a few copies in the first trimester
several hundred copies in the third trimester
Clearance of ffDNA is 48 hours after the delivery
Lo Y.M. et al. Am J Hum Genet, 1998, 62 : 768-775
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Free DNA Fetal Kit® RhD
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But, studies show that cell-free fetal DNA concentration
varies between pregnant women
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In some pregnancies insufficient fetal DNA might be
obtained
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according to known data this occurs in 0.2% of all pregnancies
In some pregnancies the amount of fetal DNA can be very
high
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E.g. if the child is suffering from a CMV infection
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IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
By using the "Free DNA Fetal Kit® RhD"
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The detection is proceeded via PCR amplification with
three different segments of the RHD genes
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A very early detection of the fetal RHD genotyping is possible
starting with the 12th gestational week
In some rare cases even earlier
Exon 5, 7 and 10
This allows the greatest possible coverage of all RhD
variants
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IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Further aspects for fetal genotyping
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Restrict the use of antenatal Anti-D
prophylaxis
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To be given only to RhD neg women carrying a
RHD positive fetus
For only about 60% of pregnant women
For the monitoring of pregnancies at risk
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RhD – negative pregnant women with natural anti-D
Assess whether the fetus is at risk of HDFN
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IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Facts:
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In the 17 European countries:
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approximately 4`400`000 pregnancies every year
650`000 are from RhD negative women
30-40 % of them have an RhD negative fetus.
Why not continue the old way?
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RhD immunoglobulin is becoming increasingly expensive
Many women today receive unnecessary injections
of human blood products
Human material is a precious source
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Population 1/1/2009
Birth 2008
15% RhD- mothers
Germany
82 002 356
682 514
102 377
France
62 448 977
796 044
119 407
UK
61 634 599
794 383
119 157
Italy
60 045 068
575 810
86 372
Spain
45 828 172
518 917
77 838
Netherland
16 485 787
184 669
27 700
Greece
11 260 402
115 500
17 325
Belgium
10 750 000
124 991
18 749
Portugal
10 627 250
104 594
15 689
Sweden
9 256 347
109 301
16 395
Austria
8 355 260
77 752
11 663
Switzerland
7 701 856
76 900
11 535
Denmark
5 511 451
65 038
9 756
Finland
5 326 314
59 530
8 930
Ireland
4 450 014
74 819
11 223
Luxembourg
493 500
5 596
839
Monaco
32 543
296
44
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IMMUNOHEMATOLOGY
Method and Kit Content
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
What is this kit about?
Bio-Rad Laboratories
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A non invasive fetal RHD genotyping of plasma DNA from
RhD - negative pregnant women
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Legal manufactured by Institut de Biotechnologie Jacques Boy
under the label of Bio-Rad
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Using Real Time PCR
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Determination of the RHD status of the fetus through maternal
blood, by a non invasive blood sampling
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Allows to prevent the risk of fetal anemia and haemolysis
when the mother is serologically RhD neg and the fetus is
RHD pos
IMMUNOHEMATOLOGY
Method
Blood samples
centrifugation
Plasma
DNA extraction
Plasma DNA
PCR amplification
Fetal RHD sequences detected
yes
Fetal genotype :
RHD positive
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IMMUNOHEMATOLOGY
no
Fetal genotype:
RHD negative
Free DNA Fetal Kit RhD
PRODUCT
1 Kit = 87 tests
REF number 060001
RHD positive (+) Control:
6 x 1000 µL (red top)
RHD negative (-) Control:
6 x 1000 µL (green top)
[100X] Maize DNA Control (not ready to use):
3 x 14 µL (yellow cap insert)
Maize exon IVR2 primers sense/antisense + probe:
3 x 38 µL (green cap insert)
RHD exon 5 primers sense/antisense + probe:
3 x 38 µL (purple cap insert)
RHD exon 7 primers sense/antisense + probe:
3 x 38 µL (white cap insert)
RHD exon 10 primers sense/antisense + probe:
3 x 38 µL (red cap insert)
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Additional equipment and accessories
Extraction
• QIAamp DSP Virus Kit (IVD CE) 50 columns, QIAGEN ref. 60704
Amplification
• Thermocycler Bio-Rad Dx Real-Time System (IVD CE) ref. 94000 centrifuge and microplate
or
• LightCycler® Roche and LC Carousel Centrifuge 2.0 Roche
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Dx Real-Time System Bio-Rad
LightCycler® Roche:
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IMMUNOHEMATOLOGY
RHD and RHDΨ
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
RH Locus
RHD-positive individuals may have one or two copies of RHD
RHD
gene
RHCE
gene
RhD-positive individuals
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RhD-negative individuals
RH gene
The antigens of the Rh system are encoded by a
pair of paralogous genes on chromosome 1
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RHD
RHCE
These genes each have 10 exons
They share 94% of its sequence identity
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RHD neg and RHDΨ
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Today we know there are several genetic causes for the RhD-negative
phenotype.
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Caucasians:
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In serologically RhD negative individuals the RHD gene is nearly always absent
Black Africans:
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only 18% of serologically D-negative black Africans are homozygous for an
RHD deletion
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66% of D-negative black Africans have an inactive RHD gene, called RHDΨ
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RHDΨ does not produce any D epitopes
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IMMUNOHEMATOLOGY
RHDΨ
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RHD genes producing variant D antigens should give a positive result
and this is being achieved by exons 7 and 10
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But - exons 7 and 10 are not suitable for testing any population
containing people of African origin, as they will give false-positive
results when the fetus has RHDΨ
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A testing including exon 5 will give a negative reaction with RHDΨ
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Exon 7 and 10 will amplify the pseudogene RHDΨ
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When the mother has the RHDΨ, an early positive reaction is given by
the maternal DNA
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IMMUNOHEMATOLOGY
Amplification Curve DΨ
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IMMUNOHEMATOLOGY
How to discriminate between fetal
and maternal DNA?
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The RHD genotyping is performed only in plasma
from RhD – negative phenotyped pregnant women
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It is expected that any RHD positive result is from
the fetus
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Separation of fetal DNA from maternal DNA has
remained unachievable
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The mothers RHD DNA is amplified at about
32-34 Cq
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The RHD DNA from the fetus is amplified after
35 Cq at about 37- 38 Cq
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Interpretation
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The analysis is interpretable if:
Test validation according to controls
The RhD negative (-) Control, and the blank control:
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No amplification detected with exon 5, 7 and 10
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The RhD positive (+) Control:
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Amplification signals for exon 5, 7 and 10
(Cq constantly < 38 cycles)
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Amplification signals for Maize DNA Control
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(internal control of extraction)
allows to validate
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the efficiency of the extraction
the absence of PCR inhibitors for each tested sample
If Maize Cp is > 36: repeat the test
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IMMUNOHEMATOLOGY
The analysis is interpretable if:
Patient interpretation
The positive results will present a value of Cp between 34 and 40
cycles
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The awaited values of Cp could be rather different depending on
PCR Instrument used.
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varying according to the gestational week
A validation for the Cycler in use must be performed
The results with Cp beyond 40 cycles are not to be considered as
definitive, they must be checked
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IMMUNOHEMATOLOGY
Interpretation
If discordant results between the three PCR amplifications
are observed it can be due to:
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a problem of a technical issues
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a problem of sensitivity difference between the three exons due to
the presence of a very small DNA quantity
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Exon 5 is the most sensitive, then exon 7 and 10
A none coding or coding variant for the fetal RhD antigen which can
be identified later by the RhD phenotyping of the newborn
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IMMUNOHEMATOLOGY
Cq > 35
(fetus)
Cq < 35
(mother)
Exon
7
10
5
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Positive
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DΨ, DV, DBS, DVI
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Negative (always control on a 2nd blood sample)
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Negative (DHAR)
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Positive (DIV)
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dCes, DBT
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RHD silent maternal gene (ser. neg)
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Positive
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DΨ, DV, DBS, DVI
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Positive (DIV)
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dCes, DBT
C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
RHD Genotype
Interpretation
If there is any doubt – give the
pregnant woman the RhD
prophylaxis!
C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Internal fetal DNA control marker
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Internal fetal DNA control marker
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Several strategies have been proposed to confirm
the presence of fetal DNA in the maternal plasma,
in the following slides some of them will be shortly
described
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Internal fetal DNA control marker
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WHO has made an international WHO standard by diluting
freeze-dried plasma of an RhD-positive man in plasma of
an RhD-negative woman
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This human DNA is not from a fetus and therefore is not
suitable to use as an internal control
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Maternal cell-free DNA in plasma of pregnant women
consists of longer fragments than in non pregnant women
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Fetal fragments are much shorter than maternal fragments
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IMMUNOHEMATOLOGY
Internal fetal DNA control marker
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The most widely used fetal DNA marker is a
Y chromosome-specific sequence
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This is just applicable if the fetus is a boy
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IMMUNOHEMATOLOGY
Internal fetal DNA control marker
Hyper- and Hypomethylation
The promoter of the RASSF1A gene is:
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hypermethylated in fetal DNA
hypomethylated in maternal DNA
It is discussed as not being a good candidate for
universal internal control for fetal DNA
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The technique is time-consuming
Exhibits too low sensitivity and specificity compared to the fetal
RHD genotyping
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Maize as an internal control
Exogenous DNA (maize) is provided as:
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Extraction/amplification control because an universal control for fetal
DNA is not yet available
Amplification of the maize DNA added to each plasma and
control provides a control for:
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adequate DNA extraction
PCR amplification
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Maize as an internal control
Exon 7
Exon 10
Maize
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Exon 5
Results from Evaluation
and Studies
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IMMUNOHEMATOLOGY
Results from Evaluation and
Studies
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In France more than 5000 fetal RHD genotypings from
maternal blood have been performed
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the results obtained confirmed the procedure of the Free
DNA Fetal Kit® RhD
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Results from Evaluation and
Studies
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A study concerning 300 samples pregnant RhD negative
women was made by using Exon 7 and 10
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The results were compared with the phenotype RhD of
the child after the birth
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100 % of correlations were obtained between the 2
methods
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However, a false negative result cannot be excluded in
the absence of a universal fetal DNA marker
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C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Results from Evaluation and
Studies
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A second retrospective evaluation of 120 plasma
samples was performed using the Free Fetal DNA Kit®
RhD (by using exon 5, 7 and 10)
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These samples were from pregnant woman having an
RHD-negative phenotype,
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five were carriers of a silence gene.
The results were compared to the phenotypes of the children at
birth with a correlation of 100%.
C. Rouillac-Le Sciellour et al. in the CNRHP (Paris-France)
Bio-Rad Laboratories
IMMUNOHEMATOLOGY
Free DNA Fetal Kit® RhD
Conclusion
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Earliest detection of cell-free DNA from
plasma of RhD negative women
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No impact on pregnancy
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High specificity due to real-time PCR test
method
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Standardized and reliable
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Avoids unnecessary prophylaxis
treatments
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Primers for RHD Exon 5, 7 and 10
The Free DNA Fetal Kit® RhD is CE marked
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Thank you for your attention
QUESTIONS?
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